The isolation or separation of white blood cells and, especially, lymphocytes from human blood is clinically necessary for histocompatability determinations, particularly in those instances of patients requiring organ transplants. Determinations of lymphocyte function are indicated where the type and level of medication needed for immunosuppression are at issue.
The present method for isolating lymphocytes and monocytes from anticoagulated human blood drawn by conventional phlebotomy techniques involves bouyant density centrifugation of cells through a particular newtonian fluid, customarily Ficoll-Paque.RTM., a liquid having a specific gravity of 1.077 g/cc and being marketed by Pharmacia Fine Chemicals AB, Uppsala, Sweden. The method contemplates the following four general steps:
(1) a predetermined amount of Ficoll-Paque.RTM. is dispensed into the bottom of a test tube;
(2) a sample of whole or diluted blood is carefully pipetted onto the Ficoll-Paque.RTM.;
(3) the Ficoll-Paque.RTM.-blood preparation is then centrifuged at about 400-500 G's for about 30-40 minutes such that the blood constituents having a specific gravity greater than the Ficoll-Paque.RTM. (1.077) pass into and/or through that liquid; and, thereafter,
(4) the white cells (predominantly lymphocytes) are pipetted off the Ficoll-Paque.RTM. phase.
This method has several disadvantages:
First, if, during the initial pipetting, white cells are accidentally deployed below the surface of the Ficoll-Paque.RTM. medium, the reduced specific gravity of the "local" Ficoll-Paque.RTM. is inadequate to separate the lymphocytes and monocytes.
Second, if, during centrifugation, lighter phases in the blood are carried into the Ficoll-Paque.RTM. medium, they cannot ascend therethrough because of the low buoyant force produced by the 400-500 G's.
Third, centrifugation forces greater than about 400-500 G's cannot be utilized inasmuch as Ficoll-Paque.RTM. is water soluble and greater centrifugation speeds increase the solubility thereof in blood, thereby resulting in a change in its specific gravity.
Fourth, after the centrifugation is completed, the pipetting of the white cells off the Ficoll-Paque.RTM. medium must be undertaken with substantial care because of the newtonian character of the medium.
Fifth, the method requires one-to-two hours for completion--a more rapid process would be highly desirable.
The determination of platelet counts in human blood samples is very important clinically for determining deficiencies in blood clotting. The separation of the platelets from the other blood constituents, particularly the erythrocytes and leucocytes, would make such counting much more rapid. Moreover, in certain types of hemophilia the lack of platelets leads to severe bleeding. One of the procedures which has been devised to counteract this problem has been to administer platelet-enriched plasma at the site of the bleeding to aid in clotting.
The present method for isolating platelets involves drawing blood into an anticoagulated tube and then subjecting to centrifugation. Most of the erythocytes and leucocytes settle to the bottom of the tube leaving plasma with suspended platelets in the upper layer. The platelet-containing plasma is thereafter carefully pipetted off in an attempt to avoid contamination from the red cells.
As is apparent, the method requires the very tedious step of carefully pipetting off the plasma fraction but, even more importantly, by its very nature the separation of platelets from the red and white cells is not nearly complete.
In certain clinical determinations the presence of platelets obscures the function of lymphocytes and/or monocytes such that their essentially complete separation from the latter two types of cells becomes vital.